Figure 2: BAG3 knockdown desensitizes C2C12 cells to matrix stiffness.
A) Schematic illustration of the SPAAC in situ stiffening reaction, which takes place upon light irradiation if the initial formulation contains excess DBCO groups. B) Young’s modulus (E’) measurement of a SPAAC hydrogel with rheology. Hydrogel was composed of a DBCO:N3 stoichiometric ratio of 4:1, and irradiated with 365 nm light with 5 mW/cm2 intensity for 120 s with 2 mM LAP. Normalized YAP/TAZ Nuc:Cyto ratio of (C) shNT and (D) shBAG3 cells cultured on stiff (E’= 32 kPa), soft (E’ = 4 kPa), and in situ stiffened (E’ = 4 → 32 kPa) hydrogels. In situ stiffening was carried out 24 h after seeding. *** < 0.001 in a one-way ANOVA test comparing means obtained on E’ = 4 and 32 kPa hydrogels, and ### < 0.001 in a one-way ANOVA test comparing means w.r.t to 24 h post-seeding timepoint. n > 155 cells, 3 independent hydrogels. E) Proposed mechanism for BAG3 regulation of YAP/TAZ mechanotransduction.